Photonic Crystal Enhanced Microscopy (PCEM) is a label-free biosensor-based form of microscopy that has been used for quantitative, dynamic imaging of cell-surface interactions. See W. Chen, K. D. Long, M. Lu, V. Chaudhery, H. Yu, J. S. Choi, J. Polans, Y. Zhuo, B. A. Harley, and B. T. Cunningham, “Photonic crystal enhanced microscopy for imaging of live cell adhesion,” Analyst, vol. 138, pp. 5886-94, 2013 and Y. Zhuo, J. S. Choi, T. Marin, H. Yu, B. A. Harley, and B. T. Cunningham, “Quantitative Imaging of Cell Membrane-associated Effective Mass Density Using Photonic Crystal Enhanced Microscopy (PCEM),” Progress in Quantum Electronics, vol. 50, pp. 1-18, 2016. The PCEM technique uses a nanostructured photonic crystal optical resonator as a substrate for cell attachment. Engagement of cell membrane components with the surface of the photonic crystal (PC) results in highly localized shifts in the resonant reflected wavelength from the biosensor. It was found that by using a modified bright field microscope cell-surface attachment could be visualized with a 0.6 μm×0.6 μm pixel resolution. The PC nanostructure interacts with broadband external illumination from a light emitting diode (LED) to establish an electromagnetic standing wave (an evanescent field) that extends only about 200 nm into the cell media. This PCEM approach is only responsive to cell membrane components that reside within the evanescent field, with the greatest response obtained for dielectric material that displaces the media closest to the PCT surface. As reported, PCEM images were acquired in time steps of about 30 seconds, thereby enabling live cell attachment “movies” to be developed. Because PCEM is label-free, it does not require photobleachable or cytotoxic reporters, enabling cells to be studied over extended time periods (up to days or weeks) to observe, for example, apoptosis, chemotaxis, and differentiation.
This previously reported cell imaging by PCEM uses an imaging modality in which the Peak Wavelength Value (PWV) of the resonant reflected peak is measured over the imaging field of view to derive images of Peak Wavelength Shift (PWS) that occur when cells attach to the PC surface. It was found that sequences of PWS images or “movies” clearly show the evolution of cell attachment through engagement of the lipid bilayer membrane and internal cell-associated proteins within the ˜200 nm deep evanescent field region of the PC.
Focal Adhesions (FAs), or cell-matrix adhesions, are large specialized protein assemblies (including mechanosensing, cytoskeletal, and signaling proteins) typically located at the interface between the cell membrane and the extracellular matrix (ECM). FAs are critical for supporting cell membrane structure and for regulating signal transmission between the actin cytoskeleton and the transmembrane receptor integrins during adhesion and migration. New tools are needed for studying the dynamic behavior of FA clusters and their interaction with the ECM, which are fundamental to processes that include metastasis, apoptosis, and chemotaxis. The response of FA clusters to drugs is one approach by which the action of pharmaceutical compounds may be evaluated, where approaches that enable characterization to be performed with a small number of cells is especially valuable. During the dynamic assembly/disassembly of a FA, the size of the FA cluster varies, and is highly correlated with the level of adhesion engagement and migration speed. For example, non-mature Focal Complexes (FXs) are initially formed at the leading edge of the cell (e.g. in the lamellipodia area) and are smaller than 0.2 μm2. As the lamellipodia withdraws from the leading edge, many FXs disassemble and release adhesion proteins back to the inner cell body, while some of the FXs grow larger (typically 1-10 μm2) and assemble into mature FA clusters by recruiting adapter proteins. Once the remaining FAs are in place, they may form stationary attachment points by binding to the ECM. A cell may utilize such anchors to migrate over the ECM through pushing/pulling the whole cellular body.
The detailed mechanism of FA assembly/disassembly in live cells, including FA dimension variation, is poorly understood, although a variety of approaches have been utilized to investigate their mechanisms. Determining the dynamic dimension of a FA cluster (with all of the FA proteins simultaneously) is challenging, especially during the assembly/disassembly process in live cells. Fluorescent tags are typically used to mark individual focal adhesion proteins, but due to the temporal limitations imposed by photobleaching, accurate quantitation and long-term analysis are exceedingly difficult to perform. In addition, cytotoxicity of fluorescent tags can compromise the viability of the cells under study.
Accordingly, there is a need to provide label-free modalities that can be used to dynamically track focal adhesions and other cellular structures, processes, and interactions.